1. Interferogram and real-time acquisition methods
Dean visit June 2008
12/10/08
Interferogram and real-time acquisition methods
Peter Kiraly
NMR Methodology Group,
School of Chemistry, University of Manchester
Workshop on Pure Shift NMR
12th Sep 2017

2. Outline Why are we interested in decoupling?
Basic concepts in pure shift NMR
Active and passive spins
Active spin refocusing elements: BS / Zangger-Sterk / PSYCHE / BIRD
The interferogram acquisition method
Theory and pulse sequences
Illustrative examples
The real-time acquisition method
Summary

3. Suppressing multiplet structure – heteronuclear decoupling11 16 16 6 7 11 15 15 7 6
proton
coupled
13C
proton
decoupled
13C-{1H}
Partial 13C NMR spectrum of estradiol in DMSO-d6 with (bottom) and without (top) proton decoupling

4. Suppressing multiplet structure – homonuclear decoupling
1H NMR of glucose in D2O
pure shift
conventional 1H
Partial 1H NMR spectrum of glucose in D2O

5. The concepts of spin decoupling
time / s
0.5 1
1.5 2 1H
Heteronuclear
decoupling
WALTZ / GARP /
adiabatic bi-level ?
Homonuclear
decoupling
pure shift
time / s
0.5 1
1.5 2 1H
time management
selectivity problem

6. Active and passive spins in pure shift NMR
conventional 1H
50 mM quinine in dmso-d6

7. ASR - Active Spin Refocusing - elements
Magn. Reson. Chem. 35, 9 (1997)
J. Magn. Reson.
124, 486 (1997)
Angew. Chem. Int. Ed.
53, 6990 (2014)
Chem. Phys. Lett.
93, 504 (1982) BS
Zangger-Sterk
PSYCHE
180°
90° 1H
13C/15N
BIRD
Small flip angle
CHIRP pulses
frequency selective
RSNOB pulse
frequency selective
RSNOB pulse
180°
180° 1H 1H 1H Gz Gz
all work by differentially manipulating active and passive subpopulations of protons

8. BS Zangger-Sterk PSYCHE BIRD
ASR - Active Spin Refocusing - elements
Magn. Reson. Chem. 35, 9 (1997)
J. Magn. Reson.
124, 486 (1997)
Angew. Chem. Int. Ed.
53, 6990 (2014)
Chem. Phys. Lett.
93, 504 (1982) BS
Zangger-Sterk
PSYCHE
180°
90° 1H
13C/15N
BIRD
Small flip angle
CHIRP pulses
multiple frequency RSNOB pulse
multiple frequency RSNOB pulse
180°
180° 1H 1H 1H Gz Gz
all work by differentially manipulating active and passive subpopulations of protons

9. active spin refocusing 180 °pulse
Double spin echo experiment with an ASR element
active spin refocusing 180 °pulse
excitation
90 °pulse
broadband
180 °pulse
41
chunk
acquisition
chemical shift is refocused at the beginning of acquisition, but …
… homonuclear coupling (J) is refocused 21 later
J-evolution is negligible during the first few data points (period 41) delivering a small chunk of the desired pure shift FID

10. Interferogram pure shift experiment – 2D acquisition
pure shift FID
analogous to 2D NMR
0 / sw1
0 / sw1 1H Gz
J-refocus
J << δ allows
data acquisition
in ‘chunks’ of
duration 1/sw1
0.5 / sw1
0.5 / sw1 1H Gz
J-refocus
t1/2
t1/2 1H
J-refocus Gz

11. active spin refocusing 180 °pulse
The interferogram pure shift pulse sequence
drop points delay
active spin refocusing 180 °pulse
excitation
90 °pulse
broadband
180 °pulse
chunk
acquisition
Chemical shift is refocused  = [integer] / sw later than the beginning of acquisition
J is refocused at the midpoint of each chunk (41)
Incremented delay allows recording all chunks of pure shift FID (during t1  evolves, but J is refocused)
Phase cycle and/or PFG pairs can enforce CTP selection

12. active spin refocusing 180 °pulse
The triple spin echo analogue with an ASR element
drop points delay
active spin refocusing 180 °pulse
excitation
90 °pulse
broadband
180 °pulse
broadband
180 °pulse
chunk
acquisition
Frequency-swept broadband pulses can be implemented
Useful to deal with strong coupling artefacts e.g. in TSE-PSYCHE
Duration of the chunk is not limited by duration of gradient pulses, but more relaxation loss

13. Resolution in interferogram pure shift experiments
duration of the complete interferogram
(pure shift acquisition time)
number of
chunks
line-width at half height
3.8 Hz 16
0.3 s
2.2 Hz 32
0.6 s
1.5 Hz 64
1.2 s
Interferogram band-selective pure shift spectra of an AX spin system calculated in Matlab/Spinach.
(20 ms chunk duration; JAX = 10 Hz)

14. Effect of duration of chunk (SW1)
pure shift
peak height
side-bands
peak height
duration of chunk
1/SW1 (SW1)
number of chunks
(TD1)
25 Hz
94 %
<4 %
40 ms
(25 Hz) 32
50 Hz
20 ms
(50 Hz)
<1 %
99 % 64
<0.2 %
100 Hz
100 %
10 ms
(100 Hz)
128
Interferogram band-selective pure shift spectra of an AX spin system calculated in Matlab/Spinach.
(AQ = 1.3 s; JAX = 10 Hz)

15. Effect of RF phase instability (scan-to-scan)
irreproducibility
±0°
analogous to t1-noise in 2D NMR
±1°
interleaved
2D data acquisition
is advisable for long experiments
±3°
±5°
Interferogram band-selective pure shift spectra of an AX spin system calculated in Matlab/Spinach.
(AQ = 1.3 s; JAX = 10 Hz; SW1 = 50 Hz)

16. Speeding things up real-time approach interferogram approach
J. Magn. Reson.
124, 486 (1997)
Pure shift FID
a set of experiments
1 single experiment
1st experiment
J. Magn. Reson.
218, 141 (2012)
2nd experiment
3rd experiment
4th experiment

17. Prototype pulse sequence of real-time pure shift NMR
Chunks are collected from a single scan
Resolution is worse compared to interferogram experiment, because of
relaxation, diffusion/convection, and cumulative errors of pulse imperfection

18. Relaxation losses in real-time experiments[ ]
HSQC
sequence elements
acquisition
J-refocus
acquisition n r r
line-width at half height
Selected traces of real-time pure shift HSQC BIRD.
The total echo time (r) of the J-refocusing element was incremented.
Broadening here is due to proton T2 relaxation.

19. Effect of BIRD timing error
If  = 1 / (2 * 1JCH), then BIRD element refocuses the 13C-attached protons.
Selected traces of real-time pure shift HSQC BIRD (1CH = 190 Hz).
The echo time () of the BIRD element was varied (HSQC sequence element was kept constant).
Broadening here is due to BIRD timing error.

20. Effect of pulse imperfection in real-time pure shift HSQC using BIRD
Gradient pulses omitted,
basic phase cycle,
and no supercycle…
G1-4 varied chunk-to-chunk
extended phase cycle (*2),
and MLEV-16 supercycle…
2nd carbon 180 pulse is omitted in BIRD…
… extra signals next to the pure shift signal and F1 mirror images
… extra signals appear due to heteronuclear
J-evolution during 2
… clean

21. 15N HSQC of L80C mutant N-PGK protein in water (90%H2O / 10%D2O)
pure shift 15N HSQC
N-terminal domain of PGK
J. Biomol. NMR. 62, 43 (2015)

22. 10 mg amikacin in D2O
pure shift 13C HSQC
13C HSQC

23. Mixture of glucose, trehalose, raffinose, and -cyclodextrin
13C HSQC
pure shift 13C HSQC

24. Interferogram vs real-time pure shift NMR
experiment time
0.5 min
conventional 1H
5 min 30 s
interferogram
Zangger-Sterk
real-time Zangger-Sterk
0.5 min
50 mM quinine in dmso-d6

25. Summary Interferogram experiments Real-time acquisition
Resolution enhancement requires extended experiment time
There is a trade-off between experiment time and quality of spectrum
Many kinds of active spin refocusing element are available
System instability may affect very high resolution experiments
Real-time acquisition
Experiment times of parent and pure shift methods are practically identical
Simultaneous sensitivity and resolution enhancement in real-time HSQC using BIRD
Resolution enhancement is limited by relaxation, diffusion/convection, and pulse imperfection
Lock disturbance can be a problem in high resolution experiments

26. Acknowledgements
Gareth Morris, Mathias Nilsson
and the NMR Methodology Group
Funding

27. A Pure Shift NMR Workshop
11.00 Gareth Morris Welcome, introduction and history
11.30 Peter Kiraly Interferogram and real-time acquisition methods
12.00 Laura Castañar Zangger-Sterk and band-selective methods
12.30 Mohammadali Foroozandeh PSYCHE
Lunch and poster session
14.00 Ralph Adams Other pure shift and related methods
14.30 Mathias Nilsson Practical implementations
15.00 Adolfo Botana JEOL pure shift implementation
15.10 Vadim Zorin MestreNova pure shift implementation
15.20 Ēriks Kupče Bruker shaped pulse implementation
Question and answer session
University of Manchester, 12th September 2017